1. Technical Field
The present disclosure relates to a chip with micro-channel which is formed on a laminated substrate of silicon and plastic. More particularly, the present disclosure relates to a chip with micro-channel which integrally has functionalities for quickly and conveniently extracting and amplifying desired DNA from an analyte containing a gene, or detecting a sequence of the DNA.
2. Related Art
In recent years, progress of genetic diversity analysis and expression analysis has been outstanding owing to improvement of DNA analysis techniques. Particularly in medical fields, the relationship between diseases and genes attracts attention. For example, by analyzing information of individual genetic information (specific DNA sequence) related to a disease, suitable treatment or administration can be carried out for each individual patient (personalized medicine). In personalized medicine, in situ diagnostics are most desirable, and speedy and convenient techniques with a strong aspect of POCT (Point of Care Testing) are desired. Therefore, it is strongly desired to realize a device capable of quickly and conveniently extracting and amplifying DNA of a gene to be analyzed from a collected analyte such as blood, and detecting a sequence of the DNA.
As one of means to meet these requirements, micro-total analysis systems (μTAS) (also called as a lab-on-chip) have attracted attention in recent years. In the μTAS or lab-on-chip, micro-channels and ports having fine structures in a micrometer order are provided in a substrate, and various kinds of operations including mixing, extraction, refinement, chemical reaction and/or analysis of a substance, and so on can be performed within the structures. The μTAS has been partially put into practical use. Since various kinds of operations are performed within fine structures, the μTAS has the following features compared to the same type of device in common size: (1) the use amounts of a sample and a reagent are remarkably small; (2) the analysis time is short; (3) the sensitivity is high; (4) it can be carried to an actual spot to perform analysis on the spot; and (5) it is disposable. Structures prepared for the purpose described above and having fine structures such as micro-channels and ports in a substrate are collectively called as a chip with micro-channel or device with micro-fluid.
For analyzing DNA in a gene in an analyte in a short time using a chip with micro-channel, it is necessary to incorporate functionalities of extraction and amplification into the chip, and realization of a fine filter for separating impurities such as blood cells and a PCR (polymerase chain reaction) capable of increasing and decreasing the temperature at a high speed is required. In addition, convenience in use is required, and it is therefore desirable to be able to stably retain an analyte, a reagent and the like in the chip. Further, in personalized medicine applications, it is desirable to have a configuration that allows treatment from blood, and to be able to sense a single base-multiple system (SNP) in DNA at a detection section. That is, it is desired to realize a versatile chip that can flexibly adapt to operating conditions.
However, due to limitations on the nature of a material of a substrate that forms a chip with micro-channel, it is difficult to realize a device with micro-channel which can meet all the foregoing requirements. The reason for this will be described below.
Plastic or silicon is used as a material of a substrate of a chip with micro-channel. The plastic substrate has such a feature that material costs are relatively low, it is easy to perform cutting processing, and affinity with a biological/bio material is relatively high, so that a reagent is easily retained, and so on. On the other hand, however, the plastic substrate has a problem that it is not suitable for formation of a fine filter structure for separating impurities such as blood cells and for formation of a thermal reactor for which it is required to increase and decrease the temperature at a high speed, such as a PCR (polymerase chain reaction), because it is difficult to process fine structures in a sub-micrometer order and the thermal conductivity of the material is not satisfactory. The silicon substrate is suitable for formation of a fine filter structure and a PCR thermal reactor because fine structures are easily formed by a semiconductor lithography technique and the thermal conductivity is higher by 2 to 3 order of magnitude than that of plastic. On the other hand, however, there is the problem that the unit price of the material is high in comparison with plastic, and the silicon substrate is not suitable for storage of a reagent because affinity between the surface of silicon and a biological/bio material is not necessarily high, and therefore non-specific adsorption of a protein and DNA occurs. As described above, plastic and silicon have mutually contradictory advantages and disadvantages, and with a configuration using a substrate of only one of silicon and plastic, conditions required for a chip with micro-channel for used in DNA analysis cannot be adequately satisfied.
As means for solving the above-described problems, a chip with micro-channel has been proposed in which a silicon substrate and a plastic substrate are laminated (see, for example, BioMed Microdevice (2011), 13:19-27 and Proceeding of 43rd International Symposium on Microelectronics (IMAPS 2010), 000036).
In BioMed Microdevice (2011), 13:19-27, a structure is disclosed in which plastic and silicon are laminated, and a chip with micro-channel and a liquid delivery section are arranged separately. In this method, however, the temperature cannot be increased and decreased at a high speed because a thermal reactor is formed of a plastic material, and also the reactor is isolated from silicon as a heating surface with a glass substrate interposed therebetween, so that it is very difficult to secure a thermal contact, leading to poor heat conduction. Further, the method cannot be used conveniently because it employs a structure in which an analyte and a reagent are supplied from outside the chip. Therefore, the method has the problem that it is not suitable for quick and convenient treatments.
In Proceeding of 43rd International Symposium on Microelectronics (IMAPS 2010), 000036, a structure is disclosed in which a thermal reactor is formed in a silicon chip, and an analyte and a reagent are supplied from the inside of the chip. Therefore, increasing and decreasing of temperature at a high speed and convenient treatments can be achieved. However, in this method, a sensor is confined to a DNA micro-array chip, and this is formed on a silicon substrate identical to that of the thermal reactor (PCR). That is, steps of producing the thermal reactor and the sensor should be successively performed, and the design cannot be flexibly changed according to an intended purpose. Further, only one thermal reactor is mounted, and only a refined genome can be used as an analyte, so that a treatment from blood cannot be performed. The method cannot adapt to applications that require two stages of PCRs: a PCR intended for extracting a genome to be analyzed from blood and a PCR intended for selectively amplifying DNA based on presence/absence of a SNP in the object to be analyzed. Further, a filter for separating and removing blood-derived blood cells generated during the treatment is not present. That is, the method has the problem that it cannot adapt to applications of detection from blood, and is therefore poor in versatility.
That is, the disclosed methods have the major problem that both a configuration of a high-performance chip with micro-channel which is necessary to perform DNA analysis quickly and conveniently and a configuration of a chip with micro-channel which has high versatility cannot be achieved.
The present disclosure has been made for solving the problems described above. One non-limiting and exemplary embodiment provide a DNA chip with micro-channel for DNA analysis, which performs extraction/amplification of DNA or detection of a sequence of the DNA quickly and conveniently and which has high versatility.